Biometric variability of inflorescence and flower traits among ex situ accessions of the neotropical oilseed palm Acrocomia Mart.

Abstract The oilseed palm genus Acrocomia is suitable for sustainable oil production in South America. The high phenotypic diversity of wild populations poses a challenge for the delimitation of the genus. Comparing the inflorescence architecture, a first‐order panicle, and staminate and pistillate flower traits could be a valuable tool in resolving the taxonomic disarray. Thus, this study aims to characterize the differences in the inflorescence architecture and floral structures of three common and economically significant Acrocomia species: A. aculeata, A. totai, and A. intumescens. Biometric traits of the inflorescence architecture and floral structures of various Acrocomia accessions in an ex situ germplasm collection in Brazil were assessed. The unweighted pair group method with arithmetic mean (UPGMA) cluster analysis based on the Gower distance was used to measure dissimilarities between the individual plants of the accessions. To our best knowledge, this study provides the first evidence of the presence of second‐order rachillae in the genus Acrocomia. Evaluated traits showed a high level of variation within and between accessions, emphasizing the phenotypic diversity of the genus. The accessions of A. totai were distinguishable from those of the other two species by their inflorescence architecture and flower traits. The dissimilarities between A. aculeata and A. intumescens were not sufficient to differentiate both. In conclusion, the quantitative assessment of inflorescence and floral traits is a valuable tool for taxonomic resolution of the genus.

The Acrocomia palms are known under a range of common names depending on the region: e.g., macaúba, bocaíuva, macaíba, or grugru in Brazil; mbocayá in Argentina and Paraguay; coyol in Mexico to Costa Rica; and corozo or tamaco in Columbia.The three arborescent species Acrocomia aculeata (Jacq.)Lodd.ex Mart., Acrocomia totai Mart., and Acrocomia intumescens Drude are of great economic interest for vegetable oil production due to their wide geographical distribution (Colombo et al., 2018), and the quantity and quality of oil produced (Freitas de Lima et al., 2021;Montoya et al., 2016;Motoike et al., 2013;Pires et al., 2013).
There has been much disagreement among scientists on the taxonomic status of the Acrocomia species and the existing literature has not distinguished between species in a systematic way.For instance, A. totai and A. intumescens are considered either synonyms (Henderson, 1995;Lanes et al., 2015;Menezes Oliveira et al., 2013;Montoya et al., 2016) or ecotypes (De Lima et al., 2020;Machado et al., 2015;Madeira et al., 2024) of A. aculeata, or as distinct species (Díaz et al., 2021;Silva et al., 2020;Vianna et al., 2021;Vianna, Berton, et al., 2017;Vianna, Carmelo-Guerreiro, et al., 2017).This contributes to a limited understanding of the economic potential of the species (de Lima et al., 2018;Vianna et al., 2021) and constraints in domestication programs and diversity conservation strategies (Hey et al., 2003;Morrison et al., 2009).Regarding the species uncertainty and a genus in need of taxonomic revision, we suggest considering the current taxonomic Acrocomia species as hypothetical taxon entities, to be confirmed or rejected by morphoanatomical and genetic data available in the present and future.
The knowledge on the structural variation of inflorescences, flower clusters, and flowers is scarce (Vargas-Carpintero et al., 2021) and focuses mainly on A. aculeata (Mazzottini-dos-Santos et al., 2015), and remains limited for other Acrocomia species and intraspecific populations.Acrocomia palms are synchronously monoecious and protogynous, that is, the pistillate flowers are receptive before the anthesis of the staminate flowers (Mazzottini-dos-Santos et al., 2015;Scariot et al., 1991).The inflorescence, an interfoliar panicle with branching of the first order, consists of a rachis carrying several hundred rachillae.The rachillae are extended and bear each distally a large number of staminate flowers present in pairs, flower clusters referred to as dyads, or solitary (Henderson, 1995;Mazzottini-dos-Santos et al., 2015;Scariot et al., 1991).At the base of the rachillae, the few pistillate flowers are represented in triads, a floral cluster of a central pistillate flower flanked by two staminate flowers (Henderson, 1995;Mazzottini-dos-Santos et al., 2015;Scariot et al., 1991).The floral morphology of the pistillate and staminate flowers shows a pronounced dimorphism.The globose pistillate flowers, with connate and overlapping petals, are larger than the elongated trimerous staminate flowers (Mazzottini-dos-Santos et al., 2015).
A thorough morphological characterization of the inflorescences and floral structures on the species and population level would facilitate an understanding of yield formation, serve as a basis for domestication initiatives, and provide important information for taxonomic, molecular, genomic, ecological, and bioeconomic studies.

| Study site and accessions of botanical material
This study was conducted at the living Acrocomia spp.Germplasm Bank, named BAG-Macaúba (register number 084/2013 SECEX/ CGEN), of the University of Viçosa, Brazil.This ex situ collection of around 300 Acrocomia populations originating from various regions of South America is located in Araponga (latitude 20°40′1′′ S; longitude 42°31′15′′ W) in the South-east of the federal state Minas Gerais in Brazil.The climate in Araponga, located at 1200 m above mean sea level, is subtropical with a dry winter and a warm summer (Cwb) according to the Köppen Climate Classification (Alvares et al., 2013).The natural vegetation is Atlantic rainforest (in Portuguese, Mata Atlântica).
Within the Acrocomia spp.Germplasm Bank BAG-Macaúba, we selected accessions of wild populations (hereafter only referred to as accessions) based on the following criteria: (A) their region of origin using the Global Positioning System (GPS) data provided by the BAG-Macaúba database; (B) the presence of inflorescence or fruit bunches in November 2018 to ensure producing palms; (C) the number of plants per accession in the collection (at least 3); and (D) a palm height below 7 m for labor safety and time.Finally, we retained six accessions with a total of 31 palms (Table 1).
We assessed visually the vegetative traits of the stem (stem swelling, leaf base persistence, and spine coverage; Figure 1), leaves, and fruit size to assign the species to the accessions using the taxonomic key of Lorenzi et al. (2010) in combination with the biogeographic origin of the accessions.Hereafter, we refer to each accession with their species' epithet, abbreviated as ACL, TOT, and INT, for A. aculeata, A. totai, and A. intumescens, respectively, followed by their accession number (BAG-ID) in the BAG-Macaúba (see Table 1).

| Evaluated traits and the study time frame
We evaluated a total of nine morphological characteristics descrip- fruit bunches were detached from the palms by severing the peduncle with a cutting device.The total length of the rachillae was determined with a tape measure.Furthermore, it was noted if the staminate flowers already started anthesis at the time of sampling.In addition, the lengths of the apical region, containing only the staminate flowers, and the basal region, containing the pistillate and staminate dyads, were measured.Fresh and dry weights of each rachilla, pistillate flower, and the total bulk of staminate flowers per rachilla were determined.All botanical materials were dried at 65°C in a ventilated oven to constant weight.Lastly, the dried staminate flowers were counted.

| Inflorescence and flower traits
After the harvest of the fruit bunches, the length of the rachis was measured with a measuring tape.The rachillae were separated from the rachis with pruning shears.The rachillae of the individual fruit bunches were grouped according to the number of ramifications and then counted.

| Statistics and picture editing
The inflorescence architecture and flower traits were statistically described with the following measures of position and dispersion: Median, interquartile ranges (IQRs), probability density, and median absolute deviation (MAD).
Spearman's rank correlation coefficient was used to assess the association between the following traits: rachis length, number of rachillae, rachillae dry weight, rachillae length, length of proportions of rachillae with pistillate and staminate flowers, number of pistillate and staminate flowers per rachillae, and dry weight, height and diameter of pistillate flowers.
All descriptive measures and Spearman's rank correlation coefficients were calculated using JMP 17 software, Version 17.1.0(JMP Statistical Discovery LLC, USA).To evaluate the shared inflorescence architecture and flower traits between the individual palm trees, we subjected the traits to cluster analysis using the Gower distance and the UPGMA algorithm.The analyses were performed using the distance and cluster procedure in SAS software, Version 9.4 (SAS Institute Inc., USA).All pictures were edited using Luminar 4 software, Version 4.3.5 (Skylum Software, USA) to correct exposure and contrast across the entire image.In the picture g in Figure 2 and the pictures in Figure 3, the background (a white tile floor) was edited out using Affinity Photo, Version 2.3.1 (Serif (Europe) Ltd., UK).No changes in tint, tone, shade, and color hue were made in all pictures.

| Inflorescence architecture and rachillae biometry
While emerging interfoliar, the young inflorescences were enclosed by a large woody peduncular bract.The bracts split abaxially along longitudinal slits, releasing inflorescences with an intense sweet odor.The abaxial face of the persistent bracts was covered in a dense layer of short brown trichomes with the occasional aculeus (Figure 2d-e).Their adaxial surface was bright yellow and glabrous (Figure 2f).
The inflorescences of all palms were consistent in their structure, being composed of a main rachis and shorter first-order rachillae, bearing the flowers.The rachis length was positively correlated with the number of rachillae per inflorescence (r s = 0.66, p < .0001; Figure 4).The inflorescences of TOT266 and TOT301 were visually distinguishable from the inflorescences of the ACL and INT accessions (Figure 2a-b).The shorter rachis (Figure 5a) with the lower number of rachillae (Figure 5b) were suberect between the leaves.The short rachillae (Table 2) gave the panicle a more rigid appearance.The inflorescences of INT123 were visually not discriminable from ACL125 and ACL267.They had a comparable length of the rachis and number of rachillae.The panicle had a pendulous appearance with the long rachillae dangling from the rachis.

| Floral biometry
The rachillae displayed an androgynous nature with an overabundance of staminate flowers in comparison to pistillate flowers with a ratio of staminate:pistillate flowers from 51:1 to 133:1 (Table 3).
The number of staminate flowers varied between 68 and 666, with some differences observed between the accessions.ACL267, INT123, and ACL125 possessed a higher number of staminate flowers compared to TOT266 and TOT301, which was attributed to the elongated apical region of the rachillae documented in the former three accessions.The Spearman correlation analysis also revealed a significant positive correlation between the number of staminate flowers and the length of the rachillae (r s = 0.78, p < .0001)and the proportion of length bearing the staminate flowers (r s = 0.68, p < .0001).
ACL267, TOT266, and TOT301 exhibited the greatest range in the number of pistillate flowers per rachilla.Rachillae with only staminate flowers and no pistillate flowers were prevalent in TOT266 and TOT301, and somewhat frequent in ACL125, mainly in the apical third of the inflorescences (Figure A1).A common trait shared by all palms was the decrease in the number of staminate and pistillate flowers, as well as the length of the rachillae, from the base to the apex of the inflorescence (Figure A1, Tables A1 and A2, respectively, for a number of pistillate flowers, number of staminate flowers, and length of rachillae).
Visually, the pistillate flowers of TOT266 and TOT301 were distinguishable from those of INT123, ACL267, and ACL125 due to their size (Table 4), shape, and color.The pistillate flowers of TOT266 and TOT301 were small, globose, and yellow-greenish in

| Sequence and frequency of floral structures
The floral structures along the rachillae are displayed in Figure 6, showcasing the sequence from the basal end to the first single staminate flower at the apical end.The studied rachillae generally followed the sequence of floral structures as described for Acrocomia-bearing triads at their basal end and staminate flowers Notably, several unusual variations were observed.For instance, dyads of pistillate flowers (Figure 2i) were found in 28 out of 132 rachillae assessed in ACL267 (Table 5a).In TOT266 and INT123, dyads of pistillate flowers were also present but a rare occurrence.
Staminate flowers were present not only as dyads or flanking the pistillate flowers in triads, but also as singlets and in triplets (Figure 6).
Only TOT266 and TOT301 displayed staminate flowers with a welldeveloped infertile carpel (Table 5b and Figure 2k).This variation was observed in the triads and staminate dyads, and at the apical end of individual staminate flowers.
The most noteworthy deviation was the occurrence of secondorder rachillae in A. totai (Table 5c and Figure 3).These rachillae were observed in the most basal positions of the first-order branches, preceding the pistillate flowers of the rachilla.While a single secondorder rachilla was the most common, up to 6 were counted.The second-order rachillae were sunken into depressions in the firstorder branch in a similar manner to the flowers.The second-order rachillae displayed the usual pattern observed in Acrocomia rachillae, with single staminate flowers at the tip and pistillate flowers at the base, although 77.4% of the second-order rachillae (data not shown) were not bearing any pistillate flowers.

| Cluster analysis
The evaluation of the accessions revealed species-specific morpho- A differentiation of TOT266 and TOT301 was not possible.

| DISCUSS ION
The delimitation of the species A. aculeata, A. intumescens, and A.
totai relies heavily on morphological characteristics of the stem and fruits and the information on geographical distribution.The identification of the species is hindered by overlapping characteristics and a high phenotypic diversity of the intraspecific wild populations.
In addition, hybridization of species can occur in areas where they coexist (Díaz et al., 2021), particularly between the widely distrib- ture oscillations, water availability, and high vapor pressure deficit (Brancalion et al., 2018;Göldel et al., 2015;Hill et al., 2023;Souza et al., 2023).We observed floral groups reduced from triads by abortion of either pistillate or staminate flowers, i.e., solitary staminate or pistillate flowers and staminate dyads in between the triads.
The most surprising observation, though, was the presence of second-order rachillae.As far as we know, this study is the first to document the existence of second-order rachillae in Acrocomia species.These second-order rachillae were only discovered in certain individual palms of A. totai accessions, in particular TOT266, which emphasizes not only the phenotypic diversity of the genus but also the phenotypic variation within wild populations.
In their comprehensive study on floral structures in A. aculeata,  (Adam et al., 2005(Adam et al., , 2007)).The mantled phenotype affects both genders where fertile or sterile androecia are transformed into carpel-like structures (Adam et al., 2005(Adam et al., , 2007) ) resulting in sterile staminate flowers with carpels and partially or totally sterile pistillate flowers with supernumerary carpels (Adam et al., 2005;Beulé et al., 2011).A phenotype expressing similar structurally abnormal flowers was found in date palm (Cohen et al., 2004).These phenotypes are observed in oil palm and date palm plants regenerated through micropropagation by somatic embryogenesis (Beulé et al., 2011;Cohen et al., 2004).In both crops, these vestigial phenotypes are present in pistillate flowers, which leads to a reduction in yield.In contrast, the staminate flowers with a well-developed infertile carpel occur spontaneously in wild populations of Acrocomia.

Mazzottini
The causes of this phenotype in wild populations of Acrocomia are unknown, and pistillate flowers with supernumerary carpels are yet to be observed in Acrocomia.Further studies on the underlying  TA B L E 2 Biometric characteristics of the rachillae.
three-dimensional (3D) geometry of an Acrocomia inflorescence is characterized by the length of the rachis and the number and length of rachillae.A third important trait, not considered in this study, is the branching angle (Harder & Prusinkiewicz, 2013).The investigated accessions of A. totai presented a shorter rachis length and rachillae length, and a lower number of rachillae than those of A. Since the accessions in the current study are grown at the same site and exposed to similar environmental conditions and management, we hypothesize that the observed discrepancy may be caused by the evolutionary adaption of different reproductive strategies including pollinator attraction strategies and reproductive success, to the environmental conditions of their natural habitat.However, the complexity of plant-environment interactions and their impact on the phenotypic biometry and genetic variability Acrocomia are poorly understood.Some traits contributing to reproductive success, such as fruit size, appear to be more environmentally susceptible than the number of producing infructescences, for instance (Coelho et al., 2017).Furthermore, it remains uncertain whether the traits of any accession cause trade-offs that impact pollinator attraction and reproductive success, particularly since inflorescence architecture and flower display play crucial roles in pollinator-plant interactions (Harder & Prusinkiewicz, 2013;Prusinkiewicz et al., 2007).
The Acrocomia totai and ACL267 accessions have a high number of pistillate flowers, which may decrease the chance of each flower being visited by pollinators (Kettle et al., 2011).However, the shorter rachillae and densely arranged pistillate flowers could mitigate this effect.Additionally, it is unclear if the smaller overall inflorescence display of Acrocomia totai accessions, with their less visible bracts, adversely affects pollinator attraction (Harder et al., 2004;Harder & Prusinkiewicz, 2013), potentially resulting in reduced pollination success in plantations where different Acrocomia species are cultivated together.Fruit set in Acrocomia aculeata and Acrocomia totai is generally low and varies widely among infructescences (Scariot et al., 1995;Silva et al., 2020).In Acrocomia aculeata, no relationship was found between the number of pistillate flowers, the number of TA B L E 4 Biometry of pistillate flowers.
rachillae, the number of fruits produced, indicating that female reproductive success is majorly dependent on the flowering synchronicity of the inflorescences and the fruit maturation period (Scariot et al., 1995).Since Acrocomia fruit maturation occurs supra-annually and takes 12 to 14 months, the fruit set is influenced by factors such as seed predation and adverse climate conditions, rather than just pollination success (Montoya et al., 2016;Scariot et al., 1995).
Despite variations in biometrics among the six accessions, the inflorescences consistently displayed traits associated with beetle pollination.These traits include yellow to cream-colored inflorescences with small staminate and pistillate flowers, short-lived staminate flowers clustered at the apical end of the rachillae, protogyny, and the absence of nectaries (Henderson, 1986).The primary pollinators of Acrocomia are derelomine flower weevils (Andranthobius spp., Derelomini, Curculionidae) and mystropine sap beetles (Mystrops spp., Mystropini, Nitidulidae;Carreño-Barrera et al., 2021;Scariot et al., 1991), both associated with a wide range of neotropical palms as a result of co-evolutionary processes (Henderson, 1986).These pollinators are present in high numbers on Acrocomia inflorescences upon bract opening and remain until the staminate flowers wither (Carreño-Barrera et al., 2021;Scariot et al., 1991) The petals of the Acrocomia flowers contain bract opening (Maia et al., 2018(Maia et al., , 2020)).The short period during which the pistillate flowers are receptive does not provide food rewards for insects.However, the apical spatial arrangement and the anthesis of the staminate flowers offer potential oviposition sites, shelter, and pollen rewards to pollinators (Carreño-Barrera et al., 2021;Henderson, 1986;Scariot et al., 1991).The characteristics of the inflorescences and flowers also attract the cyclocephaline scarab beetles (Cyclocephala spp., Melolonthidae, Cyclocephalini; Gonçalves et al., 2020;Maia et al., 2020).These beetles are considered pests in Acrocomia, as they feed on flower tissue and cause pistillate flower abortion (Oliveira & Ávila, 2011).
However, the extent of yield damage caused by Cyclocephala spp.
is currently unknown.Future research into insect-plant dynamics, coupled with the recognition of phenotypic distinctions within the genus, is crucial for understanding these ecological interactions and establishing feasible integrated pest management strategies for Acrocomia plantations.
The divergence in biometric traits found in this study supports the hypotheses of a taxonomic distinction of A. totai as an individual taxon as previously demonstrated by the geographic distribution (de Lima et al., 2018;Lorenzi et al., 2010), fruit biometry (Madeira et al., 2024;Vianna, Berton, et al., 2017), leaf anatomy (Vianna, Carmelo-Guerreiro, et al., 2017), and genetic structure of the genus (De Lima et al., 2020;Díaz et al., 2021;Lanes et al., 2015;Madeira et al., 2024).In contrast, A. intumescens could not be delimited from A. aculeata based on the inflorescence architecture and flower traits.
Besides its geographic restriction to the North-East of Brazil, the swelling of the trunk is the only morphological characteristic used to distinguish A. intumescens from A. aculeata (Díaz et al., 2021;Lorenzi et al., 2010).However, the swelling of the trunk is highly dependent on environmental factors (Tomlinson, 1990).The attempts of previous studies to delimit A. intumescens showed an overlap with A.
aculeata in fruit biometry (Vianna, Berton, et al., 2017), leaf morphoanatomy (Vianna, Carmelo-Guerreiro, et al., 2017), and genetic structure (Díaz et al., 2021).Furthermore, information on the morphological diversity of wild populations of A. intumescens is scarce and most studies conducted on the species focus on fruit biometry and oil composition.et al., 2020;Díaz et al., 2021;Lanes et al., 2015;Madeira et al., 2024;Vianna, Berton, et al., 2017;Vianna, Carmelo-Guerreiro, et al., 2017) may indicate an ongoing or established speciation process.The seed The species shows a high potential for economic exploitation based on its productivity and oil composition (Barbosa da Silva et al., 2021;Bora & Rocha, 2004).So, it is a valuable resource for domestication initiatives and breeding programs and for agronomic expansion of Acrocomia cultivation to the North-East of Brazil.

F I G U R E 7
The unweighted pair group method with arithmetic mean (UPGMA) dendrogram estimated according to the Gower distance for nine quantitative inflorescence and flower traits.Low = minimum measured, High = maximum measured.
Though not endangered taxa, Acrocomia occurs mainly in areas with high anthropogenic disturbance, and its natural habitats are highly fragmented, which may decrease the genetic diversity of the natural populations as gene flow between populations is restricted (Clement et al., 2005;Coelho et al., 2018;Navarro-Cascante et al., 2023).This is particularly the case as the major pollinators are small-bodied insects with short flight distances (Lanes et al., 2015).This makes conservation efforts crucial for preserving Acrocomia's genetic diversity (Abreu et al., 2012), considering its ecological and agronomic value.Given the environmental impact on the phenotypical plasticity of Acrocomia (Ciconini et al., 2013;Coelho et al., 2018;Machado et al., 2015), Therefore, we aimed to characterize interspecies and intraspecies phenotypical differences in the inflorescence architecture and floral structures ofA.aculeata, A. totai, and A. intumescens.The study sought to answer the following questions: (1) How do the species differ in their quantitatively measurable inflorescence and flower traits?(2) Are vestigial floral structures present, and in which species are they occurring?(3) Can the characterization of floral and inflorescence traits contribute to the taxonomic resolution between these three Acrocomia species?We assessed six accessions of wild populations located in an Acrocomia ex situ conservation collection for their inflorescence, rachillae, and flower biometry.To our best knowledge, this study is the first to report the presence of secondorder rachillae in the genus Acrocomia and to give a detailed insight into the variation of inflorescence architecture present between populations of Acrocomia.
tive for inflorescence architecture (length of rachis; number of rachillae; and length of rachillae), rachillae (dry weight of rachillae), and flower traits (number of pistillate flowers; number of staminate flowers; height of pistillate flowers; diameter of pistillate flowers; and dry weight of pistillate flowers).The length of rachillae, and rachillae and flower traits were measured during the flowering periods of 2019 and 2021.In 2020, a detailed sampling for the evaluation of rachillae and flower traits was not possible due to travel restrictions during the COVID-19 pandemic.Additionally, the number of inflorescences per palm tree was counted and labeled during the flowering periods of 2019, 2020, and 2021.As the assessment of rachis length and number of rachillae was unfeasible during the flowering periods, these measurements were taken after harvesting the fruit bunches in 2021 and 2022.The TA B L E 1 List of the six accessions of Acrocomia assessed in the Macaúba Active Germplasm Bank (BAG-Macaúba).The designation consists of the species epithet and accession number in the BAG-Macaúba.The regions of origin are deposited in the BAG-Macaúba database and the Köppen climate was excerpted from the supplemental material of Alvares et al. (2013).
Every other day during the flowering periods of 2019 and 2021, we surveyed the 31 individuals for any open inflorescences and sampled a total of nine rachillae from each newly opened inflorescence.Three rachillae were collected, respectively, from the basal, middle, and apical third of the inflorescences.To assess the variation in floral structures, we recorded for each rachilla the number of the following structures: triads and number of flanking staminate flowers present; staminate and pistillate dyads; staminate flowers with a well-developed, infertile carpel; rachilla ramifications; and triple and single staminate flowers only when present between the pistillate flowers.In addition, the order of occurrence of these floral structures was determined from the most basal end of the rachilla to the apex.The most basal floral structure was assigned "Position 1" and the character of the floral structure was recorded.The subsequent floral structure was designated "Position 2" and its character was also recorded.This was repeated for each subsequent floral structure until the singular staminate flowers of the apex were reached.Afterward, all floral structures were separated from the rachillae.The height and diameter of the pistillate flowers were determined with a digital sliding scale.The diameter of the pistillate flowers was measured along the axis parallel to the stalk (Diameter 1) and the axis perpendicular to the stalk (Diameter 2).

F
Left side: Stem morphology of Acrocomia intumescens, A. aculeata, and A. totai.The morphology of the stem is currently the most important trait for species distinction.Right side: One plant of each accession (including the height (soil to crown base) of that specific plant in December 2018).
color.In comparison, the pistillate flowers of A. intumescens and A. aculeata were egg-shaped, larger, and heavier with a light-yellow hue.The number of pistillate flowers showed a weak negative correlation with the pistillate flower dry weight (r s = −0.12,p < .0001), the pistillate flower height (r s = −0.11,p < .0001),and the diameter (r s = −0.12,p < .0001).INT123 and ACL125 had a wider spacing of pistillate flowers within the rachillae, which was related to longer rachillae and fewer pistillate flowers.While ACL267 had similar numbers of pistillate flowers to TOT266 and TOT301, it also exhibited wider spacing along with a higher proportion of rachillae length-bearing pistillate flowers.

F
An overview of the inflorescence and floral structure of Acrocomia species.(a) Inflorescence of A. aculeata.(b) Inflorescence of A. totai.(c) Detailed view of the rachis and attached rachillae (A.aculeata).(d-f) Details of the bract showing the abaxial dense layer of short brown trichomes with the occasional aculeus, and bright yellow and glabrous adaxial surface.(g) Rachillae.(h) Triad.(i) Pistillate dyad.(j) Staminate dyad.(k) Staminate flowers with a well-developed infertile carpel in the apical end of rachillae.toward the tip (Figure 2g-j).However, some deviations from this pattern were observed, such as dyads between pistillate flowers and the absence of pistillate flowers altogether, not uncommon for Arecoideae.
logical and biometric variation of the inflorescences and floral structures.Cluster analysis based on all individual flowering palm trees divided the accessions into two groups (Figure7), one composed of solely the accessions of A. totai and the other containing the accessions of A. aculeata and A. intumescens.The division into two groups reflects the differentiation of inflorescence architecture and flower traits.The first group, characterized by traits associated with A. aculeata and A. intumescens, includes long rachises, a high number of heavy and long rachillae, and a low number of large pistillate flowers.On the contrary, the second group, associated with A. totai, exhibits short rachises, fewer rachillae, and a higher number of smaller pistillate flowers.The length of the rachis, the number and size of rachillae, and the size of pistillate flowers are positively correlated (Figure4).In the two-way clustering analysis, these groups are visually distinguishable by the division of colors, with one group showing high values for these traits and the other showing low values, thereby reinforcing their combined classification.
Among A. aculeata and A. intumescens, ACL125 and INT123 could not be distinguished based on their inflorescences and floral structure characteristics.F I G U R E 3 Second-order rachillae in Acrocomia totai.Right: Branches with 1 or 3 second-order rachillae.Left: Detail showing the branching of the second-order rachillae off the first-order branch before the start of the flower-bearing first-order rachillae.
uted A. aculeata and either A. totai, endemic to the South-West of Brazil, Paraguay, and Argentina, or A. intumescens, restricted to the North-East of Brazil.The framework of viewing the currently accepted species as hypothetical but distinct entities allows for a more exhaustive assessment of the biological diversity (morphoanatomical and genetic) of the wild populations and the potential species boundaries while allowing a quantitative data-driven debate on the taxonomic status(Galtier, 2019;Hey et al., 2003).The characterization of interspecies and intraspecies morphological differences in the inflorescence architecture and floral structures of Acrocomia populations could contribute to the taxonomic delimitation of the F I G U R E 4 Scatterplot matrix illustrating the pairwise relationship between the inflorescence architecture and flower traits of Acrocomia, along with the corresponding Spearman's rank correlation coefficient (rho).Statistically significant correlations (p < .05)are highlighted in bold.The histograms display the distributions.species and be an informative tool for domestication initiatives and breeding programs.Notably, the study of all 382 inflorescences studied showed floral structures consistent with those common to palm species of the subfamily Arecoideae.A denotative floral characteristic of the Arecoideae are the triads, a cluster of a central pistillate flower flanked by two staminate flowers(Tomlinson et al., 2011).The studied rachillae generally followed the sequence of floral structures as described for Acrocomia among others byDransfield et al. (2008), Mazzottini-dos-Santos et al. (2015), and Scariot et al. (1991), bearing triads at their basal end and staminate flowers toward the tip.However, differences in the sequence of floral structures at the basal end were a common observation.Floral variations on the flower or inflorescence level are frequent in the palm family and are also an indication of the high environmental susceptibility of the Arecaceae to factors, such as tempera- -dos-Santos et al. (2015) described the rare occurrence of two deviant floral structures: pairs of pistillate flowers and staminate flowers with well-developed infertile carpels.Staminate flowers with a well-developed infertile carpel, where stamens transformed into carpel-like structures, were also found in the present study in isolated staminate flowers of staminate dyads, triads, and in the apical region of the rachillae.Unlike Mazzottini-dos-Santos et al. (2015), we did not observe this vestigial phenotype in A. aculeata, but only in the accessions of A. totai.The staminate flowers with a well-developed infertile carpel resemble the mantled flower phenotype in oil palm (Mazzottini-dos-Santos et al., 2015), a somaclonal variation caused by epigenetic homeotic changes in the floral ABC model

F
I G U R E 5 Biometric characteristics of the Acrocomia inflorescence architecture.Length of rachis in cm (a).Number of rachillae (b).Median, quartiles, probability density (equal width), and median absolute deviation (MAD) of a total N of inflorescences of both flowering seasons of 2019/2020 and 2020/2021 at the BAG-Macaúba, Araponga, MG, Brazil.causes of the development of the mantled phenotypes could be of interest, as in vitro propagation of Acrocomia is necessary to facilitate breeding programs and the generation of planting material.In our field observations, the distinct visual differences between inflorescences of A. totai and those of A. aculeata and A. intumescens were unmistakable.Moreover, the application of the Gower distance analysis to inflorescence architecture and floral traits (Figure7) resulted in the separation of the accessions into two large clusters: the accessions of A. totai contrasted with those of A. aculeata and A. intumescens.The rachis and rachillae of an inflorescence are the branching scaffold providing physical support for the flowers and subsequently the fruits(Harder & Prusinkiewicz, 2013 aculeata and A. intumescens.This is consistent with the findings ofSilva et al. (2020) who compared wild populations of A. totai and A. aculeata based on 41 morphoagronomic descriptors including inflorescence and infructescence architecture.The short rachis and rachillae of A. totai lead to a more compact and rigid appearance of the inflorescences.Furthermore, the inflorescences were rarely pendulous, but more often nested in the leaf sheath of their corresponding leaf without a visible peduncle.By comparison, in both A. aculeata and A. intumescens the inflorescences drooped between the leaves, along the trunk, due to a visible peduncle.Moreover, the rachillae dangled from the rachis, resulting in loose appearing inflorescences.However, visual discrimination was not possible between the inflorescences of A. aculeata and A. intumescens.Compared to studies including inflorescence traits of A. totai(Silva et al., 2020;Vianna et al., 2021) and A. aculeata (Mazzottini-dos-Santos et al., 2015;Silva et al., 2020), for both species the length of the rachis of the inflorescences in the present study was shorter and the number of rachillae per inflorescence was lower, whereas the rachillae were longer.The discrepancy of the biometric traits, particularly in pistillate flower number and size, and number of rachillae between the accessions of Acrocomia totai (TOT266 and TOT301: high number of small-sized pistillate flowers) to the other accessions (ACL125 and INT123: low number of larger pistillate flowers), could be attributed to resource allocation and reproductive strategies(Kettle et al., 2011). Mazzottini-dos-Santos et al. (2015) observed a high variability in the biometry of Acrocomia aculeata flowering traits but could not distinguish between the environmental effect of the collection areas in northern Minas Gerais and the genetic variability.

F I G U R E 6
Sequence of floral structures along the rachillae from the basal end to the apical single staminate flower end of N rachillae assessed in both flowering seasons of 2019/2020 and 2021/2022 at the BAG-Macaúba, Araponga, MG, Brazil.specialized osmophores (Mazzottini-dos-Santos et al., 2015) that emit volatile olfactory semiochemicals attracting insects upon dispersal of Acrocomia totai populations to Paraguay and Argentina was probably facilitated by the Paraguay and Paraná rivers, originating in southwestern Brazil and flowing through Paraguay, Argentina (as Paraná River) to the Rio de la Plata between Argentina and Uruguay.Our results on the lack of a species delimitation of A. intumescens should be taken with caution as we had only one accession of A. intumescens at our disposal to do this study.We also want to emphasize the importance of further research on the morphological diversity of A. intumescens.
is essential to question whether phenotypical variability correlates with genetic diversity.The most feasible approach to conserving critical biodiversity is currently the population-based conservation efforts, considering the available knowledge on the genus Acrocomia.Notably for Acrocomia totai, and probably also for Acrocomia intumescens, the conservation of their genetic pool is essential as natural populations are threatened by the expansion of commercial plantations of Acrocomia aculeata to the South-West and North-East of Brazil(Vianna et al., 2021).Exchanging and planting identical genetic material from diverse wild populations in different Acrocomia living germplasm banks is crucial for comprehensive comparative studies.Maintaining these accessions at the same site over several years allows for studying phenotypic and annual variation under consistent edaphoclimatic and management conditions(Migicovsky et al., 2019).Simultaneously, exchanging material between germplasm banks enables comparisons of the individual accessions at different sites, providing insights into the impact of varying environmental conditions.This approach would contribute to distinguishing between environmental and genetic effects on traits, such as characteristics of the reproductive biology, to gain a better understanding of reproductive success and facilitate systematics, genetic, and ecological studies on Acrocomia's diversity.Our assessment of Acrocomia's inflorescence and flower traits showed a major biometric variability between and within accessions, including the existence of second-order branching.Despite the resulting overlap of the quantitatively assessed characteristics of the accessions, the study showed a distinction between A. totai from A. aculeata and A. intumescens, suggesting an ongoing speciation process.These findings highlight the potential usefulness of inflorescence and flower traits as an additional tool for the taxonomic resolution of these three species.Exploring a wider range of wild populations and accessions from germplasm collections in future studies would provide deeper insights into the intraspecific and interspecific biological diversity of these traits.The high phenotype variability observed provides further evidence of the importance to acknowledge the biological diversity of the wild Acrocomia populations in future studies and to correctly identify the taxonomic entity of wild populations and accessions in germplasm collections based on the knowledge present.

Relative frequency (%) of ramified rachillae grouped by N of second-order rachillae Number of second-order rachillae
(a) Frequency of occurrence of pistillate dyads and (b) staminate flowers with a well-developed infertile carpel of a total N of rachillae assessed in both flowering seasons of 2019/2020 and 2021/2022 at the BAG-Macaúba, Araponga, MG, Brazil.(c) Relative frequency of second-order rachillae of a total N of rachillae assessed in both harvest seasons of 2020/2021 and 2021/2022.Madeira et al., 2024).Furthermore, the geographic distribution of the two species overlaps in the Brazilian federal states of Ceará and the northern regions of the federal state of Bahia.The Serra da Mantiqueira, a mountain range located in the Brazilian states of São Paulo, Rio de Janeiro, and Minas Gerais, acts potentially as a geographical barrier that separates the populations of Acrocomia.
The similarities between Acrocomia aculeata populations from Minas Gerais and Acrocomia intumescens are likely caused by ongoing gene flow by seed spreading facilitated by the São Francisco River, which originates in central Minas Gerais and crosses four northeastern Brazilian states (Lanes et al., 2015; TA B L E 5 Acrocomia totai populations in Mato Grosso do Sul.The observed separation of Acrocomia totai from Acrocomia aculeata and Acrocomia intumescens accessions in this study and former studies (De Lima